(1) Field of the Invention
This invention relates to deblocking N-protected amino acids and peptides, including polypeptides.
(2) Description of the Related Art
A variety of procedures have been used for removing N-protected groups from N-protected amino acids and peptides.
Prior art documentation focusing particularly on processes for deblocking aspartame-type compounds (which is a preferred use of the instant invention) includes:
U.S. Pat. No. 4,539,147 to A. Filippini et al teaches a process for preparing .alpha.-L-aspartyl-L-phenylalanine alkyl esters from L-aspartic acid of which the amino group is protected by bonding it to a 3,5-dimethoxy-.alpha.,.alpha.-dimethhyl-benzyloxycarbonyl group. This reference teaches the removal of the protective group from the final product by the action of U.V. radiation.
In U.S. Pat. No. 3,933,781 to G. L. Bechman, reference is made to various other methods of removing N-protective groups from aspartame type products, inter alia, catalytic hydrogenation and treatment with mineral acids and bases, and within that context specifically, we have an example of the catalytic hydrogenation method.
U.S. Pat. No. 3,879,372 to W. H. J. Boesten which teaches removal of a carbobenzoxy N-protective group by hydrogenation using a palladium catalyst supported on activated carbon. Also, U.S. Pat. No. 4,613,460 to P. Casati et al teaches deprotection of carbobenzoxy protected nitrogen by hydrogenolysis with a palladium-on-carbon catalyst in a glacial acetic environment.
As evident from the foregoing, aspartame is a dipeptide, and as such it is formed with an amide bond between an activated carboxyl group of one amino acid and the amino group of another amino acid. The desired pure peptide requires protection of all other functional groups not involved in the peptide bond formation. Once the final product is isolated and purified, the process of removing the protective groups follows and procedures therefore depend on the type of protective groups used. These commonly are the ones used as N-protectors in peptide chemistry, namely the formyl, acetyl, benzoyl, substituted and unsubstituted carbobenzoxy, t-butoxycarbonyl and the hydrohalide salt groups.
A particularly preferred N-protective group is the formyl group which is used in conjunction with L-aspartic anhydride. French Patent No. 2,040,473, for example, teaches N-formyl-L-aspartic anhydride.
Now, it is very well known that the reaction to remove these N-protective groups is complicated since the splitting of peptidic bonds takes place at the same time with the formation of undesired by-products such as diketopiperazine.
Extensive research therefore has been focused not only on the various synthetic routes for the preparation of aspartame but on the various means to protect the amine group not involved in the peptide bond formation during the synthesis of aspartame and furthermore on the means to bring about de-blocking of said amine groups once the said aspartame compound has been produced and isolated.
When a formyl group is used as a blocking agent for the amine groups not involved in the peptide bond formation of the aspartame synthesis, then de-blocking involves finding a suitable procedure to deformylate the .alpha.-L-aspartyl-L-phenylalanine methyl ester sweetener which was synthesized, e.g., ##STR1## With respect to formyl protected amine groups, deformylation which is the object of the subject invention, is amply documented in the prior art.
One reference for instance, U.S. Pat. No. 4,638,081 to B. Elefante, teaches in a preferred embodiment deformylation through a process comprising use of normal hydrochloric acid in a hydroalcoholic solution under boiling conditions. Another reference, U.S. Pat. 4,642,367 to M. Finotto refers to the use of hydrazine or acylhydrazine in the endeavor to deformylate N-formyl protected .alpha.-L-aspartyl-L-phenylalanine methyl esters.
In a variant to the supra approaches, U.S. Pat. No. 4,656,304 to E. Oppici et al teaches the removal of the formyl group directly in the condensation mixture so as to avoid the isolation of N-formyl aspartame and thereby reduce production costs. This reference uses phosphoric acid and a lower alkyl alcohol which are added to the reaction mixture containing N-formyl .alpha.-L-aspartyl and .beta.-L-aspartyl-L-phenylalanine methyl ester so that the .alpha.-L-aspartyl-L-phenylalanine methyl ester is made to precipitate which is subsequently converted to the free aspartame by treatment with a base.
Other references teach yet additional methods for bringing about deformylation of formyl protected L-aspartyl-L-phenylalanine methyl esters, e.g. U.S. Pat. No. 4,021,418 to T. Takemote et al teaching the use of hydroxylamines in this process.
Disadvantages of the above processes have been pointed out, such as low yields, expensive reagents, esterification of .beta.-carboxy groups and hydrolysis of ester or peptide bonds. Furthermore, deformylation is remarkably affected by concentration and reaction temperatures, particularly when the mineral acid approach is used to bring about this desired deprotective reaction.